DNA Technology in Human Identification

Dr. Philip BehDr. G. Srivastava

Outline

Identification Classification Individualisation

Methods of Identification Development of Forensic DNA Forensic issues Interpretation of results Future

Identification

Classify – Eg. Human or not? Human – Sex? Race? Age? Identification – Nationality? Identification – Locality? Identification – Identity

(Individualisation!)

Human Identification

How do we identify an individual?

The human brain associates a name with an individual using a variety of information including physical appearance, mannerisms, voice and speech, etc.

Visual Identification

Despite the complexity and accuracy of visual identification of an individual, it is well known that mistakes are common and in some circumstances unreliable and difficult.

Unreliable visual ID

Time factors – aging, etc. Psychological and emotional factors –

poor recall Lighting – inadequate or problematic

lighting Physical changes especially in forensic

work.

Individualisation

One and only No other copy except for a genetic twin.

Other physical attributes

Ear-shapes Lip prints Retinal scans Voice patterns (?) Handwriting (under scrutiny!)

Other means of individualisation Blood

Blood groups HLA typing

DNA DNA Fingerprinting DNA Typing

Development of DNA Fingerprinting 1985, Sir Alec Jeffries, described

variable number tandem repeats (VNTR`s) and developed the technique restriction fragment length polymorphisms (RFLP)

Briefly, it used restriction enzymes to cut the regions of the DNA surrounding the VNTR’s.

History

First use in casework in the U.K. in 1985.

First commercial labs in the U.S. in 1986

Used by the FBI in the U.S. in 1988. Used in Hong Kong in early 90’s. DNA-PCR technology used in Hong

Kong in 1997.

Uniqueness

Except for identical twins the DNA of an individual is unique.

The number of different chromosomes that a child receives from parents are 246

Requirements for forensic casework Reliability of technique Reproducible results No laboratory error Security of test samples and results

DNA Polymorphisms

Sequence polymorphism….AGACTAGACATT…..….AGATTAGGCATT…..

Length polymorphism….AATGAATGAATG….….AATGAATG…..

RFLP

Restriction fragment length polymorphism

A restriction enzyme is used to cut the DNA into fragments at specific points

These fragments of different lengths are separated by an electric current. The fragments of interest are then radiolabelled by hybridisation.

RFLP

Many RFLP systems are based on change in a single nucleotide. They are said to be diallelic

Thus only two common alternative forms and three phenotypes, two homozygous and one heterozygous.

PCR Based Systems

Length Polymorphisms STR kits - Short Tandem Repeats

Sequence Polymorphisms PolyMarkers eg. DQ-alpha/A1 (HLA - DNA) Mitochondrial DNA

Automated systems now available.

STR

Short-tandem repeats Higher incidence of homozygotes, since

the system is less polymorphic Several loci can be amplified Increase discriminating power with use

of multiple probes.

Length Polymorphisms

PCR used to amplify this. Much simpler than RFLP analysis

because the DNA of interest already amplified.

Bands stained directly Trend towards fluorescent detection and

automated analysis.

Forensic use of DNA technologies Identification of small quantities of

biological samples found, e.g. blood stains, semen, saliva stains, etc.

Differentiation between origins of samples found.

Linking and/or grouping of unknown sample.

Sources of DNA

Obstacles in forensic casework Small quantity of samples to work with. Contamination of samples. Poor preservation of material from

which DNA is to be extracted. Eg. Contaminated stains, decomposition of tissues.

Complicating factors and forensic challenges Multiple contributors (sources) – mixed

sample. Differential extraction Degradation Contamination

Inhibition of enzymes Non-human DNA

DNA Extraction methods employed

Reverse Dot Process

Commonly used markers

CODIS

Combined DNA Index System 1990 as a pilot project at the FBI

Laboratory. Now in more than 100 public

laboratories

Quality and standards

DNA Advisory Board Quality assurance standards

Laboratory Validation American Society of Crime Lab Directors

Laboratory Accreditation Board European DNA Profiling Group Interpol European Working Party on DNA

Profiling

Controls

Monitoring for False Negatives False negatives arises from inhibitors.

Safeguard against false negatives Positive controls – similar physiochemical

properties and contains known DNA.

Controls Monitoring for False Positives False positives generally arises from

contamination. Safeguards against false positives

A negative control – undergoes the whole procedure, similar physiochemical properties except for genetic property.

A blind control – undergoes all extraction, purification and amplification procedures, except that it does not contain any sample material.

A no-template control – serves only for the amplification reagents and conditions. It contains every amplification reagents except DNA

Significance of results

Three possible conclusions:- 1. Exclusion – they are different. 2. Inconclusive 3. Similar

Similarity

Three possible scenarios:- 1. Sample from a common source 2. Coincidence 3. Accident (Error)

Frequency Estimate Calculations Hardy-Weinberg equilibrium

There is a predictable relationship between allele frequencies and genotype frequencies at a single locus. This is a mathematical relationship that allows for the estimation of genotype frequencies even if the genotype has not been seen in an actual population survey.

Frequency Estimate Calculations Linkage equilibrium

Defined as the steady-state condition of a population where the frequency of any multi-locus genotypic frequency is the product of each separate locus. This allows for the estimation of a DNA profile over several loci, even if the profile has not been seen in an actual population survey.

DNA evidence in Court

This posed a problem due to a hosts of poorly researched and performed work and estimate calculations.

It is now quite widely accepted and increasingly the frequencies are individualising e.g. 1 in several billions!!

Population Data

Population data is required to obtain the various frequencies of occurences.

Systems used require careful validation prior to use and also require internal and external controls for each test.

Future - Now Automation DNA databases Variant Repeats – approaches individualisation

with just one or two loci More loci and marker systems SNP’s Quality control will continue to be an area of

contention. More applications and more probes Faster automation, etc

DNA Databases

Privacy issues Quality control of data Convicted samples vs. forensic case

work samples

Ancient DNA

Made possible by the availability of PCR The term ancient DNA now covers any bulk

or trace DNA from a dead organism or parts of it, as well as extracorporeally encountered DNA of a living organism.

Therefore any DNA that has undergone autolytic or diagenetic processes or fixation is considered “aDNA”

Reference

Ancient DNA – Bernd Hermann & Susanne Hummel, Springer (1994) ISBN 0-387-94308-0

Mitochondrial DNA Advantage of having multiple copies present;

therefore higher chance of detection in degraded material compared with nuclear DNA.

Increasing use, different methodologies. About 4800 human mtDNA sequences available in

forensic databases. An observed sequence is reported as the number

of times an observed sequence is present in known databases and as such a frequency percentage is given. Possible to exclude 99%.

Mitochondrial DNA

Discriminatory powers are still poor. Similarities may be seen in several

individuals with similar maternal root. Useful in “population genetics” and in

tracing the maternal line.

Y-Chromosome Increasing interest since it was sequenced. Y-

chromosomes had about 6 of its 50 million DNA letters in palindromes.

Have also been used to trace the “male line” in population studies

Useful for working on mixed samples. May be key to “racial or regional characterisation”.

Large numbers of commercial kits now available.

Future

Low-copy numbers – risk of contamination high

Racial – regional origins. Physical attributes from DNA Psychological/behavioral attributes Genetic predisposition for drug

tolerance, for rare disorders, etc.

Recommended reading DNA Technology in Forensic Science – National Research

Council (1992) ISBN 0-309-04587-8 The Evaluation of Forensic DNA Evidence – National

Research Council (1996) ISBN 0-309-05395-1 An Introduction to Forensic DNA Analysis – K. Inman & N.

Rudin. CRC Press (1997)ISBN 0-8493-8117-7

Forensic DNA Typing – Biology and Technology Behind STR Markers - John M. Butler. Academic Press (2001) ISBN 0-12-147951-X

Recommended reading Rutty GN et al “Contamination of mortuary

instruments and work surfaces by human DNA; A significant problem in forensic practice?” Int. J Legal Med 2000; 114: 56-60

B.S. Weir “Population genetics in the forensic DNA debate.” Proc. Natl. Acad. Sci. USA Vol 89 pp 11654-11659, December 1992

John Whitfield “Y chromosome sequence completed” Nature Science Update 19 June 2003.